Radioiodination of 2,3-dimethyl-4H-furo[3,2-c ...

Applied Radiation and Isotopes 95 (2015) 153–158

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Radioiodination of 2,3-dimethyl-4H-furo[3,2-c]coumarin and biological evaluation in solid tumor bearing mice S.M. Abd Elhalim, I.T. Ibrahim n Labelled Compounds Department, Radioisotopes Production and Radioactive Sources Division, Hot Laboratories Center, Atomic Energy Authority, P.O. Box 13759, Cairo, Egypt

H I G H L I G H T S

Labeling of coumarin derivative with iodine-125. The appropriate conditions for labeling of coumarin derivative (95% yield). 125I-coumarin derivative was found to be highly localized in tumor.

art ic l e i nf o

a b s t r a c t

Article history: Received 29 June 2014 Received in revised form 17 September 2014 Accepted 18 September 2014 Available online 22 October 2014

Compound 2,3-dimethyl-4H-furo[3,2-c]coumarin is a coumarin derivative that could be labeled with 125 I. The process of labeling was started using 1 mg of the compound, 50 mg CAT oxidizing agent, 30 min as reaction time at pH with a yield about 95%. The 125I-coumarin derivative was stable for about 48 h. Radiochemical purity of the labeled compound was performed by electrophoresis and HPLC. The labeled compound was separated with purity about 95%. Tumor transplantation to produce a solid tumor in the right leg of albino mice was made by intramuscular injection of 2 106 EAC (Ehrlish acittes carcinoma cells). Biodistribution study of 125I-coumarin derivative revealed that the uptake in tumor bearing leg was over 5% at 1 h and 4 h post-injection. This uptake encourages the use of 123I-coumarin derivative in imaging of tumor sites. & 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

Keywords: Coumarin Iodine Labeling Biodistribution Solid tumor

1. Introduction Early diagnosis of a tumor is still one of the most significant problems to start treatment before metastasis. One of the routes to tumor imaging is to label anticancer drugs (antimetabolites, alkylating agents, metabolites, peptides, hormones and DNA or RNA interacting agents like quinoxalines and coumarins) (Adelstein and Kassis, 1996). Coumarin is a chemical compound which is found naturally in some plants, although it can be synthetically produced as well. It has a distinctive odor which has led people to use it as a food additive and ingredient in perfume. The chemical name for coumarin is benzopyrone. Coumarins are structural units of several natural products and feature widely in pharmacologically and biologically active compounds (O’Kennedy and Thornes, 1997). Coumarins have been synthesized by several routes including von Pechmann, Perkin, Knoevenagel, Reformatsky and Wittig reactions (von Pechmann and Duisberg, 1884; Johnson, 1942; Jones, 1967; Brufola et al., 1996; Shriner, 1942; Yavari et al., 1998). Besides functionalized coumarins

n

Corresponding author.

furocoumarins such as psoralens are photoactive drugs which are extensively used in the PUVA (psoralen plus UVA radiation) therapy for the treatment of human skin diseases (Singer and Long, 1996; Zahradnik, 1992; Murray et al., 1982). Most prominent among the biological activities associated with these photochemotherapeutic agents (psoralens, linear furocoumarins) is their ability to cross-link DNA via intercalation of the furocoumarin between the base pairs of the nucleic acid and [2þ2] photocyclo addition with the pyrimidine bases, particularly thymine (Maria et al., 2004). Based on their interaction with DNA the idea was to label one of these compounds (Hara et al., 2001).with radioiodine to image tumor sites. Synthesis of 2,3-dimethyl-4H-furo[3,2-c] coumarin and 3-phenyl-4H-furo[3,2-c] coumarin as angular furocoumarins were carried out through the Williamson reaction of 4-hydroxycoumarin with α-haloketones followed by cyclization (Jiao et al., 2003). The affinity of 2,3-dimethyl-4H-furo[3,2-c] coumarin towards DNA and the antibacterial activity were evaluated and compared with 8-methoxypsoralen (8-MOP) (Dall’Acqua et al., 1981). This study was conducted to label 2,3-dimethyl-4H-furo[3,2-c] coumarin with radioiodine [125I] (Fig. 1) and study factors affecting labeling yields. It will be extended to evaluate the radiolabeled coumarin derivative with convenient chromatographic methods.

http://dx.doi.org/10.1016/j.apradiso.2014.09.011 0969-8043/& 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

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S.M. Abd Elhalim, I.T. Ibrahim / Applied Radiation and Isotopes 95 (2015) 153–158

3.1. Oxidizing agent To conduct this experiment different amounts of CAT (10, 25, 50. 100, 150 and 200 mg) were used to obtain the optimum one. 3.2. Substrate content 125

Fig. 1. Labeling of coumarin derivative with radioiodine [ oxidizing agent.

I] using CAT as

The study also searches the ability of the [125I-compounds] to imaged an induced solid tumor in EAC bearing mice.

2. Materials and methods 2.1. Drugs and chemicals 1. Iodine-125 was purchased from Institute of Isotope Production, Belgum. 2. 2,3-dimethyl-4H-furo[3,2-c]coumarin is a gift from Faculty of Science-Damietta University, New Damietta. 3. Chloramine-T was purchased from Sigma Chemical Company, USA. 4. All other chemical reagents were of analytical grade (AR), obtained from reputable manufacturers. 5. Ehrlich ascites carcinoma (EAC) was kindly supplied from National Cancer Institute, Cairo, Egypt. Female Swiss Albino mice weighing 20–25 g were purchased from the Institute of Eye Research Cairo, Egypt. Environmental and nutritional conditions were kept constant throughout the experimental period and kept at room temperature (227 2) 1C with a 12 h on/off light schedule. Female mice were used in this study due to their susceptibility to Ehrlich ascites carcinoma more than male mice (Wally et al., 2010). Animals were kept with free access to food and water throughout the experiment. Synthesis of 2,3-dimethyl-4H-furo[3,2-c]coumarin and 3-phenyl4H-furo[3,2-c] coumarin. The synthetic route for the preparation of the furocoumarin derivative is displayed as phenol was converted in 50% yield to the 4-hydroxycoumarin by the reaction with malonic acid in the presence of phosphorous oxychloride as described in the literature by Al-Sehemi and El-Gogary (2012). Synthetic compound was evaluated by IR, NMR and mass spectroscopy. The structure of the compound was characterized by IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. 2.2. Iodine labeling 125 I-coumarin derivative was prepared by the following procedure: 10 mg coumarin derivative was dissolved in 1 ml DMF with stirring. CAT solution was prepared as 10:1 (w/w) in double distilled water then about 5–10 mL (50–100 mg CAT) was added to 100 ml coumarin derivative solution in dark colored tube and approximately 50–100 MBq 125I at room temperature. After a specified interval of time, the reaction was stopped using 0.2 N Na2S2O3 solutions to ensure that the un-reacted iodine is reduced before chromatographic analysis (Kassis et al., 2004). The yield of the reaction and the radiochemical purity were determined by paper electrophoresis. Paper electrophoresis techniques were conducted to distinguish between free iodide that move towards the anode at the top, while iodocompound persist near the point of spotting.

3. Factors affecting labeling yield There are many factors that affect labeling yield such as follows:

To investigate the minimum amount at which maximum labeling yield was obtained, different amounts of coumarin derivative (100, 300, 500, 1000, 1500 and 2000 mg) were used. 3.3. pH of the reaction The pH of the medium is one of the important factors that affect labeling yield. So the reaction medium was studied at different pH values (2, 4, 7, 9, 11) using pH Meter model 601A digital ionalyzer, Orion research, USA to distinguish the pH at which maximum labeling yield was obtained. 3.4. Reaction time Labeling reactions proceeded to different reaction times (1, 5, 10, 15, 20, 25, 30, 45 and 60 min). 3.5. Reaction temperature The reaction was carried out at different temperatures 25, 40, 50 and 70 1C. 3.6. In-vitro stability Stability was studied to determine the stability of 125I-coumarin derivative after labeling and the impact of time on that compound. The yield was measured at different time intervals (1, 4, 12, 24 and 48 h) after labeling. When one factor was studied other factors were kept constant. 3.7. Electrophoresis conditions Electrophoresis was done with EC 3000 p-series 90 programmable power and chamber supply units using cellulose acetate strips (45 cm). These stripes were moistened with 0.05 M phosphate buffer pH 7 and then introduced in the chamber. Samples were applied at a distance of 10 cm from the cathode. Standing time and applied voltage were continued for one and a half-hours. Developed strips were dried and cut into 1 cm segments, then counted by a well-type NaI scintillation counter. The radiochemical yield is calculated as the ratio of the radioactivity of the labeled product to the total radioactivity (Kassis, 2003). 125

% Radiochemical yield ¼

Peak activity of I coumarin derivative 100 Total activity

3.8. Tumor transplantation in mice Ehrlich ascites carcinoma cells (EAC) is a model for studying the biological behavior of malignant tumors and drugs assumed to produce effects at these sites (Ibrahim and Wally, 2010). A line of Ehrlich ascites carcinoma (EAC) was maintained in female Swiss Albino mice through weekly I.P transplantation of 2.5 106 tumor cells/mouse. EAC cells were obtained by needle aspiration with aseptic condition. The ascetic fluid was diluted with sterile saline so that 0.1 ml contains 2.5 106 cells counted microscopically using a hemocytometer. About 0.2 ml solution was then injected intramuscularly in the right leg to produce a solid


tumor leaving the left leg as a control in the peritoneal cavity to produce ascites tumor (Pressacco et al., 1994). 3.9. Biodistribution of

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Then fractions of 1 ml were collected separately using a fraction collector up to 30 and counted in a well-type NaI (Tl) detector connected to a single-channel analyzer.

125

I-coumarin derivative in normal mice 4.1. Electrophoresis analysis

In-vivo biodistribution studies were performed using 4 groups each comprising six mice. Each animal was injected in the tail vein with 0.2 ml solution containing 5–10 kBq of radioiodinated coumarin derivative. The mice were kept in metabolic cages for the required time. Animals were subjected cervical dislocation at the recommended time (15 min, 30 min, 1 h or 4 h) after injection. Organs or tissues of interest were removed, washed with saline, weighed and counted. Correction was made for background radiation and physical decay during the experiment. The weights of blood, bone and muscles were assumed to be 7%, 10% and 40% of the total body weight, respectively (Mester et al., 1996). 3.10. Biodistribution of bearing mice

125

I-coumarin derivative in solid tumor

A group of 24 solid tumor bearing mice was used for studying the biodistribution of the labeled drug at the selected times (15 min, 30 min, 1 h or 4 h) taking 6 mice at each time. Mice were sacrificed by cervical dislocation at various time intervals. Organs and tissues of interest were removed, washed, weighed and counted for its uptake of activity. Ascites fluid was drained and counted as a whole in a well type NaI (TI) gamma counter. The results were calculated as a percentage of injected dose (I.D) per gram tissue per body weight (Issidorides and Haddadin, 1996).

Fig. 4 illustrates the analysis of the fractions that were produced from the reaction by electrophoresis. Two main peaks were formed, one corresponding to the free iodide that moved towards the anode with 16 cm distance at the condition mentioned before. The second peak which stayed near the point of spotting was found to be identical to that of 125I-iododeoxyuridine under the same electrophoretic conditions (Korde et al., 1998). 4.2. Factors affecting labeling yield 4.2.1. Effect of oxidizing agents Results obtained in this study revealed that the electrophilic substitution of the iodonium ion [nI þ ] onto coumarin derivative molecule afforded a high radiochemical yield by utilizing CAT as an oxidizing agent (Fig. 5). It was observed that the radiochemical yield significantly increased by increasing the amount of CAT from

4. Results and discussion HPLC analysis showed that the retention times of KI, coumarin derivative and 125I-coumarin derivative were 3.0, 7.6 and 7.8 min, respectively, identified by UV absorption as shown in Fig. 2. Fig. 3 shows that retention time of 125I and 125I-coumarin derivative was the same retention times of 3.0 and 7.8 min, respectively, as that of the inactive KI and iodo-compound detected by UV. HPLC analysis of 125 I-coumarin complex was done by injection of 10 ml, after 0.22 mm Millipore filtration, into the column (RP-18. 300 3.9 mm 2, Alpha bond) and UV spectrophotometer detector (SPD-6A) adjusted to the 240 nm wavelength. The column was eluted with mobile phase (60% acetonitrile, 40% water), and the flow rate was adjusted to 1 ml/min.

Fig. 2. U.V. absorption chromatogram of the reaction mixture of substrate, CAT and KI.

Fig. 3. Radiochromatogram of the reaction mixture of substrate, CAT and Na125I.

Fig. 4. Paper electrophoresis pattern of the radioiodinated125I-coumarin.

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Fig. 5. Variation of radiochemical yield of 125I-coumarin derivative as a function of chloramine-T amount as an oxidizing agent. Mean 7SD (mean of three experiments). Reaction condition: 1 mg coumarin derivative, X mg chloramine-T, 10 mL Na125I. The reaction mixture was kept at 40 1C for 30 min.

10 μg to 50 μg (optimum content) at which maximum labeling yield was obtained. By increasing the amount of CAT above 50 μg, the yield showed no significant change. A significant decrease in the labeling yield was noted by decreasing the concentration of CAT below 50 μg that may be explained as at low concentrations of CAT, not all iodide converted to iodonium ion and thus, the yield was decreased (Walicka et al., 1998) 4.2.2. Effect of substrate amount The influence of coumarin derivative amount as a substrate on the labeling yield using CAT as an oxidizing agent is shown in Fig. 6. The increase of the amount of coumarin derivative was accompanied by a significant increase in the labeling yield, where it reached above 95% at 1 mg of coumarin derivative. Increasing the amount of coumarin derivative above 1 mg produced no significant increase in the labeling yield. Increasing the amount of starting material usually increases the total incorporation of radioiodine, since there is a minimum limit to the volume used (Wally et al., 2010). 1 mg of coumarin derivative was required to obtain maximum labeling yield, below this amount there was a significant decrease in the yield. On the other hand, using higher amounts did not significantly affect labeling yields.

Fig. 6. Variation of radiochemical yield of 125I-coumarin derivative with substrate (cumarin derivative) amount using chloramine-T as an oxidizing agent. Mean 7SD (mean of three experiments). Reaction condition: X mg coumarin derivative, 50 mg chloramine-T, 10 mL Na125I. The reaction mixture was kept at 40 1C for 30 min.

Table 1 Effect of pH of the reaction medium on the labeling yield of

125

I- coumarin.

pH value

% Labeled compound

% Free iodide

2 4 7 9 11

44.5 7 0.11 76.8 7 0.30n 93.8 7 0.30n 93.6 7 0.44n,† 81.2 7 0.20n,†

55.5 7 0.15 23.2 7 0.2 9.2 7 0.20 6.4 7 0.25 18.8 7 0.4

Values represent the mean 7 SEM n ¼6. n Significantly different from the initial values using unpaired student's t-test (P o0.05). † Significantly different from the previous values using unpaired student's t-test (P o 0.05).

4.2.3. Effect of pH As shown in Table 1, pH 7 is the optimum pH at which the maximum yield was obtained (97.3%). It was observed that at pH 4, the yield was 93.8% while at pH values 9 and 11 the yield was 93% and 81.7%, respectively. There was significant difference between all pH values of the reaction mediums. 4.2.4. Effect of reaction time Fig. 7 shows the relationship between the reaction time and the yield of 125I-coumarin derivative. Radiochemical yield was significantly increased from 30.9% to 94.8% with increasing reaction time from 1 min to 30 min. Extending the reaction time to 60 min produced no significant change of the radiochemical yield.

Fig. 7. Variation of radiochemical yield of 125I-coumarin derivative with reaction time. Mean 7 SD (mean of three experiments). Reaction condition: 1 mg coumarin derivative, 50 mg chloramine-T, 10 mL Na125I. The reaction mixture was kept at 40 1C for x min.

4.2.5. Effect of reaction temperature The influence of reaction temperature on the radiochemical yield of 125I-coumarin derivative is shown in Fig. 8. The reactions were carried out at 25, 40, 50 and 70 1C. At room temperature, the

labeling yield was 85 72.0% and increased to the maximum radiochemical yield 94.897 1.0% at 40 1C and decreased at higher temperature (70 1C) to 82.5 74.6%. This may be due to the thermal decomposition of the 125I-coumarin derivative.


4.3. In-vitro stability of

125

I-coumarin derivative

In the present experiment, no significant change in the stability of 125I-coumarin derivative up to 48 h post-labeling is observed as shown in Table 2. This result showed the stability of the labeled compound. 4.4. Biodistribution of

125

I-coumarin derivative

4.4.1. In normal mice A biodistribution study of 125I-coumarin derivative in normal mice showed that radioiodinated coumarin derivative was distributed rapidly in blood, stomach, kidney and liver at 15 min post-injection. After 30 min, 125I-coumarin derivative uptake was significantly decreased in organs like blood, liver and spleen. However, 125I-coumarin derivative uptake was significantly increased in stomach, bone, muscle, and thyroid after 30 min. At 1 h and 4 h post-injection, the majority of tissues showed significant decrease in 125I-coumarin derivative uptake. Thyroid gland showed significant increase in 125I-coumarin derivative uptake at 1 h post-injection as shown in Table 3. 4.4.2. In solid tumor bearing mice The sites of greatest uptake of 125I-coumarin derivative after 15 min post-injection were the blood, stomach, heart and lung, respectively. Table 4 shows that the concentration of 125I-coumarin derivative was the lowest in thyroid, spleen and muscle at 15 min post-injection. The uptake of 125I-coumarin derivative in solid tumor (right leg) was rapidly taking place as it received 3.5% of total activity. The uptake of solid tumor was significantly increased after 30 min and 1 h to reach 6.2% and 7.8% per g, respectively. No significant change in the uptake of 125I-coumarin derivative in solid tumor at 4 h post-injection was observed when compared to its

157

previous value. The data also showed that some organs exhibit significant increase of uptake at 30 min post-injection like the stomach, solid tumor, kidney and thyroid. On the other hand, significant decrease in 125I-coumarin derivative uptake was observed in blood, heart and lung at the same time. At 1 h post-injection, the majority of organs showed significant decrease in uptake of 125Icoumarin derivative. Significant increase in 125I-coumarin uptake was only observed in solid tumor and thyroid at 1 h post-injection. Similarly, at 4 h post-injection, the majority of organs showed additional significant decrease in 125I-coumarin derivative uptake. The results revealed that the solid tumor was the site with one of the most uptake of 125I-coumarin derivative and this was seen at 30 min and lasted to 4 h post-injection. This result suggests the use of 125I-coumarin derivative in imaging of a tumor. The high uptake of 125 I-coumarin derivative in the kidney may reflect the excretion of the drug via urine (Issidorides and Haddadin, 1996). The observation that the percentage of 125I-coumarin derivative concentration in the thyroid was significantly less than in other tissues indicates that less free iodide is associated with the 125I-coumarin derivative, since free iodide is rapidly captured by thyroid (Shriner, 1942). However, Table 3 Biodistribution of

125

I-coumarin in normal mice.

Organs and body fluids

Percent I.D./gram organ Time post-injection 15 min

30 min

1h

4h

8.4 7 0.04n 4.7 70.3n 18.6 7 0.1 16.4 7 0.2n 1.3 7 0.05 1.9 7 0.10n 1.4 7 0.10n 0.7 70.1n 1.1 7 0.01 1.7 7 0.02n 1.2 7 0.10 0.5 70.02n 4.4 7 0.05 1.8 7 0.15n 1.17 0.06n 1.0 70.02 2.7 7 0.1 5.107 0.12n 2.5 7 0.20n 1.2 70.01n 3.20 7 0.8 3.4 7 0.30n 2.0 7 0.01n 1.1 70.04n 6.2 7 0.9 10.2 7 0.60* 9.6 7 0.16n 5.7 70.2n 4.4 7 0.50 5.2 7 0.30n 3.5 7 0.1n 1.5 70.03n 6.7 7 0.4 9.2 7 0.60n 3.17 0.3n 1.5 70.06n 6.5 7 0.7 15.5 7 0.7 23.5 7 0.7 36.5 70.7 1.4 7 0.3 1.17 0.10n 1.0 7 0.02 0.8 70.05n 0.7 7 0.02 1.2 7 0.14n 4.17 0.16n 5.2 70.2

Blood Bone Muscle Liver Lung Heart Stomach Intestine Kidney Urine Spleen Thyroid

Values represent mean 7SEM. n Means significantly differ from the previous each value using unpaired student's t-test (p o0.05).

Table 4 Biodistribution of

125

Organs and body fluids

I-coumarin derivative in solid tumor bearing mice. % injected dose/organ Time post-injection 15 min

Fig. 8. Variation of radiochemical yield of 125I-coumarin derivative with reaction temperature. Mean 7 SD (mean of three experiments). Reaction condition: 1 mg coumarin derivative, 50 mg chloramine-T, 10 mL Na125I. The reaction mixture was kept at x 1C for 30 min. Table 2 In-vitro stability of

125

I-coumarin.

Time/hours

% Labeled compound

% Free iodide

1 6 12 24 48

94.17 0.36 93.7 7 0.30 93.9 7 0.40 92.8 7 0.30 92.5 7 0.30

6.9 7 0.55 7.3 7 0.2 6.17 0.1 8.2 7 0.2 7.5 7 0.30

Values represent the mean7 SEM n¼ 6.

Blood Bone Muscle Liver Lung Heart Stomach Intestine Urine Kidney Spleen Thyroid Left leg Right leg

30 min

1h n

4h n

7.7 7 0.2 3.2 7 0.2n 19.3 7 1.8 10.81 7 0.6 2.4 7 0.15 3.5 7 0.1 2.4 7 0.0n 1.17 0.1n 1.3 7 0.01 1.9 7 0.02n 1.5 7 0.10 0.7 7 0.02n 4.5 7 0.4 7.7 7 0.2n 2.17 0.1n 1.7 7 0.1n 7.5 7 0.5 1.5 7 0.3n 47 0.2n 3.2 7 0.15n 97 0.4 4.4 7 0.1n 2.7 7 0.05n 2.5 7 0.2 9.3 7 0.7 10.6 7 1n 7.5 7 0.5n 5.2 7 0.26n n n 4.17 0.2 12.7 7 0.1 14.17 0.3 3.6 7 0.25 4.5 7 0.7 12.5 7 0.7 21.5 7 0.7 34.5 7 0.7 n n 2.5 7 0.7 6.6 7 0.2 7.5 7 0.3 2.5 7 0.2n 0.7 7 0.5 1.2 7 0.4n 2.6 7 0.5n 1.4 7 0.05n 0.6 7 0.2 0.4 7 0.3n 2.17 0.5n 6.4 7 0.9n 1.4 7 0.05 1.6 7 0.1 1.5 7 0.1 1.2 7 0.03n 4.7 7 0.3 6.2 7 0.5n 7.8 7 0.2n 5.2 7 1.1n

Values represent mean 7SEM n¼ 6. n Significantly different from each previous value of each organ using unpaired student's t-test (Po 0.05).

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References

Table 5 T/NT values. Time

15 m

30 m

1h

4h

T/NT

4.7/1.4

6.2/1.6

7.8/1.5

5.2/1.1

thyroid uptake was increased with time post-injection due to in-vivo deiodination of 125I-coumarin derivative (Ibrahim and Wally, 2010; Pressacco et al., 1994). 4.4.3. Target/non-target ratio (T/NT) Regarding the T/NT it was observed that it was more than 3, 3.8, 5 and 4.75 at 15 min, 30 min, 1 h and 4 h respectively. These values could provide the ability to image tumor sites (Table 5). 5. Conclusion The incorporation of Auger emitters [125I] to a tumor site was achieved by labeling of coumarin derivative with iodine-125. The appropriate conditions for labeling of coumarin derivative (95% yield) were 50 μg CAT as oxidizing agent, 1 mg coumarin derivative as substrate at pH 7 at room temperature and 30 min reaction time. The great incorporation of 125I-coumarin derivative in tumor sites facilitates tumor imaging. 125I-coumarin derivative was found to be highly localized in tumor sites which considered an ideal vector to carry radioiodine to the nucleus of tumor cells and encourage further application to evaluate this radiolabeled compound in-vivo and in-vitro on cancer cell lines. It also encourages the use of 123I-coumarin in the diagnosis of tumor sites.

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